Sialidases are enzymes present on viral protein coats and bacterial outer membranes. They serve to process the carbohydrate moieties of glycoproteins and glycolipids terminated with sialic acids that are found on host cell surfaces. The processing of these glycoprotein and glycolipid moieties are crucial in the pathogen replication cycle. A specific example of this particular host-pathogen species is the influenza virus and the erythrocyte. Influenza virus binds to the erythrocytes through attachment to cell surface carbohydrates. Specifically, the virus has a membrane envelope with two types of surface glycoproteins, the hemagglutinin and the neuraminidase (sialidase), both of which interact with sialyloligosaccharides on host (erythrocyte) cells. It has long been known that pretreatment of erythrocytes or host cells with bacterial sialidase abolishes viral adsorption and/or infection, demonstrating that sialic acid is an essential feature of the receptor determinant (Burnet, F. M. and Stone, J. D., Aust. J. Exp. Biol. Med. Sci., 25, 227-233 (1947) and Stone, J. D., Aust. J. Exp. Biol. Med. Sci., 26, 48-64 (1948)). Hemagglutinin attaches to the host cell structure which contains sialic acid, galactose and N-acetylglucosamine. Neuraminidase functions to hydrolyze the sialic acid from receptors, and at high pathogen levels in an infected cell this neuraminidase activity aids in elution of the budding virus from the host membrane, thus facilitating replication of the pathogen.
Given the mechanism of the influenza pathogenicity, two possible routes to inhibition of infection could be theorized. One route is the inhibition of viral hemagglutinin binding to the host cell, which would deny the virus its initial foothold on the cell surface. Several studies have attempted to design molecules that would competitively inhibit viral hemagglutinin binding. Pritchett et al., Virology, 160, 502-506 (1987), teach a procedure for evaluating the relative affinities of simple sialoside receptor determinants in their interaction with the receptor binding pocket of the influenza virus hemagglutinin. Pritchett determined that siaiosides with the NeuAc.alpha.2, 6Gal and NeuAc.alpha.2, 6GlcNAc linkages were 5-30 times more potent inhibitors than those with the NeuAc.alpha.2, 3Gal linkage.
Pritchett et al., J. Biol. Chem., 264, 9850-9859 (1989) teach that sialic acid and galactose are both important components of the natural substrate for initial viral recognition.
Sabesan et al. (U.S. Pat. No. 5,220,008) disclose hepta-saccharides containing bivalent receptor trisaccharide determinants that bind the influenza virus with increased inhibitor potency. The compounds of Sabesan are two trisaccharide structures comprising sialic acid (N-acetylneuraminic acid), galactose and N-acetyl-glucosamine which are attached to another (anchoring) sugar molecule.
The second potential route to inhibition of viral infection involves the synthesis of compounds that serve as preferred binding substrates for the sialidase (typically neuraminidase) enzymes wherein these substrates could not be hydrolyzed, or would be hydrolyzed very poorly by the sialidase. Such substrates could function as competitive binding inhibitors. Attempts at inhibiting the active site of the sialidases have involved isolation of active components from bacterial cultures, plant extracts, or the production of enzyme site specific antibodies.
Frolov et al. (JP 0326248) report the isolation and partial characterization of an influenza neuraminidase inhibitor from S. aurens having a molecular weight of 94,0000-120,000 which demonstrated 100% inhibition of type A0, A1 or A2 influenza neuraminidase.
Air et al., J. Virol., 64, 5797-5803 (1990), describe monoclonal antibodies which inhibit influenza virus neuraminidase by binding to epitopes located on the rim of the enzyme active-site crater. Air evaluates neuraminidase inhibition by the monoclonal antibody NC41 from nineteen different influenza mutants and concludes that there is a high degree of strain specificity at the enzyme active site.
Nagai et al., Chem. Pharm. Bull., 38, 1329-1332 (1990), describe inhibition of influenza and mouse liver sialidases by flavonoids isolated from 103 species of plants. The inhibitory flavone compound was not fully characterized.
One of the difficulties in using the sialidase inhibitors described above as potential pharmaceuticals is that most remain generally uncharacterized and their specific mechanisms of action are unknown. Recently, carbohydrate moeities have been synthesized which have binding affinity for the sialidase active site but are not hydrolyzed. For example, Suzuki et al., Glycoconjugate, 7, 349-356 (1990), teach synthetic thioglycoside analogs of disaccharide gangliosides that competitively inhibit GM3 hydrolysis by the sialidase of different subtypes of human and animal influenza viruses.
In an effort to elucidate the three dimensional properties of possible sialidase substrates, Sabesan et al., Carbohydr. Res., 218, 27-54 (1991), examined the conformational characteristics of several synthetic sialyloligosaccharides present as terminal sequences in N and O-linked carbohydrate groups of glycoproteins. Sabesan teaches that for .alpha.- D-NeuAc-(2-6)-.beta.-D-Gal linkages the C-6 arm of the galactose can potentially orient either in the low energy "gt" or the high energy "tg" orientation. In any given population the "gt" conformer will predominate over the "tg" conformer in ratios on the order of 85%:15%.
The present invention addresses two aspects of pathogen inhibition by providing novel disaccharide compounds that are expected to bind hemagglutinin (and thus function as competitive inhibitors with the natural substrate), but are either not hydrolyzed or are poorly hydrolyzed by sialidase. The invention also provides novel monosaccharide intermediates which are useful for preparation of these disaccharides. The novel (NeuAc-Gal) disaccharides are based on the natural trisaccharide substrate, .alpha.DNeuAc(2-6).beta.DGal(1-4).beta.DGlcNAc, which is present on glycoproteins and glycolipids found on host cell membranes. Applicant has discovered that with the addition of an alky group to the C-6 arm of the galactose (which prohibits free rotation about its C-6 to C-5 bond axis and freezes the structure in a rigid conformation) it is possible to create two conformer analogues of the disaccharides, a high energy and a low energy conformer, which differ in three dimensional conformation and which possess distinctly different properties. Applicant has found that only the high energy conformer is able to selectively bind or inhibit influenza virus sialidase activity. Neither the low energy conformer or the racemic mixtures of the instant compounds are effective enzyme inhibitors. Based on the general art and the work of Pritchett et al., J. Biol. Chem., 264, 9850-9859 (1989) and Sabesan et al., (U.S. Pat. No. 5,220,008) Applicant believes the instant compounds will serve as competitive inhibitors to pathogenic hemagglutinin binding to host cells. Further, Applicant has demonstrated that the freezing of the compounds in rigid conformation results in a dramatic change in sialidase substrate activity and thereby the instant compounds serve as useful inhibitors of the sialidase activity associated with pathogenic organisms.